Robotic transanal minimally invasive surgery (TAMIS) for local excision of rectal lesions with the da Vinci Xi (dVXi): technical considerations and video vignette.

Robotic transanal minimally invasive surgery (TAMIS) (RT) represents a compelling new alternative capable of overcoming the limitations of conventional TAMIS for the local excision of rectal lesions. We describe our RT technique using the dVXi™ (Intuitive Surgical, Sunnyvale, CA, USA) which we have used to efficiently and completely excise eight cases of rectal lesions which were not endoscopically resectable. We also include a video vignette of the procedure. With the patient in the prone jackknife position, we insert a GelPOINT™ Path Transanal Access Platform (Applied Medical, Rancho Santa Margarita, CA, USA) in combination with the dVXi and AirSeal™ insufflation system (Conmed, Niagara. Falls, ON, Canada). Our technique aims to be ergonomically efficient to minimise docking difficulties and to reduce instrument clash in the limited space, whilst maximising the capabilities of the dVXi for RT. At 3-month endoscopic follow-up, no evidence of recurrence was detected in any of the eight patients. RT is safe, feasible and has advantages over conventional laparoscopic TAMIS (LT). Our described technique addresses some of the long-standing challenges of LT and the novel RT. The immediate challenge to its widespread use remains the cost, expertise and availability.

Pharmacokinetics of enrofloxacin and ceftiofur in plasma, interstitial fluid, and gastrointestinal tract of calves after subcutaneous injection, and bactericidal impacts on representative enteric bacteria.

This study's objectives were to determine intestinal antimicrobial concentrations in calves administered enrofloxacin or ceftiofur sodium subcutaneously, and their impact on representative enteric bacteria. Ultrafiltration devices were implanted in the ileum and colon of 12 steers, which received either enrofloxacin or ceftiofur sodium. Samples were collected over 48 h after drug administration for pharmacokinetic/pharmacodynamic analysis. Enterococcus faecalis or Salmonella enterica (5 × 10(5) CFU/mL of each) were exposed in vitro to peak and tail (48 h postadministration) concentrations of both drugs at each location for 24 h to determine inhibition of growth and change in MIC. Enrofloxacin had tissue penetration factors of 1.6 and 2.5 in the ileum and colon, while ciprofloxacin, an active metabolite of enrofloxacin, was less able to cross into the intestine (tissue penetration factors of 0.7 and 1.7). Ceftiofur was rapidly eliminated leading to tissue penetration factors of 0.39 and 0.25. All concentrations of enrofloxacin were bactericidal for S. enterica and significantly reduced E. faecalis. Peak ceftiofur concentration was bactericidal for S. enterica, and tail concentrations significantly reduced growth. E. faecalis experienced growth at all ceftiofur concentrations. The MICs for both organisms exposed to peak and tail concentrations of antimicrobials were unchanged at the end of the study. Enrofloxacin and ceftiofur achieved intestinal concentrations capable of reducing intestinal bacteria, yet the short exposure of ceftiofur in the intestine may select for resistant organisms.

Early uterine serous carcinoma: clonal origin of extrauterine disease.

Uterine serous carcinoma (USC) is an uncommon but aggressive type of endometrial carcinoma that is frequently associated with extrauterine disease despite minimal or no myometrial invasion. The origin of the extrauterine tumors in this setting remains controversial. The majority of USCs (90%) and endometrial intraepithelial carcinomas (78%), the putative precursor of USC, have p53 mutations, suggesting that p53 alterations occur early in the pathogenesis of USC. To determine if the extrauterine tumors associated with minimally invasive USC and endometrial intraepithelial carcinoma (EIC) represent metastases or multifocal primary tumors, we examined the mutational pattern of the p53 gene in 3 cases of minimally invasive USC and 1 case of EIC and in the corresponding extrauterine tumors associated with each of the cases. In all 4 cases, the primary tumors and the associated extrauterine tumor foci had identical p53 mutations. Our results support the premise that extrauterine serous tumors found in association with EIC or minimally invasive USC represent a unifocal process and thus are early metastases.

Resolution of structural isomers of sialylated oligosaccharides by capillary electrophoresis.

The resolution of structural isomers in mixtures of oligosaccharides is often challenging. Capillary electrophoresis was employed to separate three sets of structural isomers of sialylated oligosaccharides found in human milk and bovine colostrum. Different running buffers were necessary to achieve optimal baseline resolution. To resolve 3'- and 6'-sialyllactoses, 0.2 M aqueous sodium phosphate containing 40% methanol as an organic modifier was used as a running buffer. To resolve 3'- and 6'-sialyllactosamines, 0.4 M aqueous sodium phosphate without organic modifier was used. Baseline resolution of sialyllacto-N-tetraose-a and -b and sialyllacto-N-neotetraose-c was achieved with a 0.4 M Tris-HCl buffer containing 250 mM sodium dodecyl sulfate and 10% methanol as the organic modifier. Thus, each of these sets of structural isomers of sialylated oligosaccharides required a unique running buffer with respect to buffer type, concentration, pH, presence of organic modifiers, and surfactants. Similar electrophoresis conditions may be useful for resolving and analyzing other structural isomers of acidic oligosaccharides by capillary electrophoresis.

Fucosylated human milk oligosaccharides vary between individuals and over the course of lactation.

Specific human milk oligosaccharides, especially fucosylated neutral oligosaccharides, protect infants against specific microbial pathogens. To study the concentrations of individual neutral oligosaccharides during lactation, a total of 84 milk samples were obtained from 12 women at 7 time periods during weeks 1-49 postpartum. The neutral oligosaccharides from each sample were isolated, perbenzoylated, resolved, and quantified by reversed-phase high-performance liquid chromatography. The resultant oligosaccharide peaks, identified by co-elution with authentic standards and mass spectrometry, ranged in size from tri- to octasaccharides. The total concentration of oligosaccharides declined over the course of lactation; the mean concentration at 1 year was less than half that in the first few weeks postpartum. One of the 12 donors produced milk fucosyloligosaccharides that were essentially devoid of alpha1,2 linkages (but contained alpha1,3- and alpha1,4-linked fucose) until late in lactation, consistent with the nonsecretor phenotype. In milk samples from the remaining 11 donors, fucosyloligosaccharides containing alpha1,2-linked fucose were prevalent, and their profiles were distinct from those of fucosyloligosaccharides devoid of alpha1,2-linked fucose. The ratio of alpha1,2-linked oligosaccharide concentrations to oligosaccharides devoid of alpha1,2-linked fucose changed during the first year of lactation from 5:1 to 1:1. Furthermore, the absolute and the relative concentrations of individual oligosaccharides varied substantially, both between individual donors and over the course of lactation for each individual. The patterns of milk oligosaccharides among individuals suggest the existence of many genotype subpopulations. This variation in individual oligosaccharide concentrations suggests that the protective activities of human milk could also vary among individuals and during lactation.

Comparison of oligosaccharides in milk specimens from humans and twelve other species.

Human milk contains large amounts of many oligosaccharides, most of which are fucosylated; several inhibit pathogenic bacteria, viruses, and toxins that cause disease in humans. Although bovine milk is known to have much less and many fewer types of oligosaccharides, no studies heretofore have indicated whether the amount or complexity of human milk oligosaccharides is unique to our species. Toward this end, a comparison was made of the major individual oligosaccharides in milk specimens from a variety of species, including the great apes. The neutral compounds, which represent the bulk of oligosaccharides in human milk, were isolated, perbenzoylated, resolved by high performance liquid chromatography (HPLC), and detected at 229nm. Ambiguous structures were determined by mass spectrometry. All milk specimens contained lactose, although levels were quite low in bear and kangaroo milk. The types of oligosaccharides in milk specimens from the primates resembled those of human milk, but the amounts, especially of the larger molecules, were markedly lower. The relative amounts of oligosaccharides in the bonobo changed over the course of lactation, as they do in humans. Marine mammals generally had few oligosaccharides in their milk other than 2'-fucosyllactose. Grizzly and black bear milk specimens contained a wide range of oligosaccharides, many of which had novel, fucosylated structures. Milk specimens from humans, bears, and marsupials had the greatest quantity of, and the most complex, neutral oligosaccharides. Although human milk contained more oligosaccharide than did milk specimens from the other species studied, the presence of appreciable amounts of complex oligosaccharides was not unique to humans. This finding suggests that in animal milk specimens, as in human milk, neutral fucosylated oligosaccharides potentially offer protection from pathogens to offspring with immature immune systems.

Survival of human milk oligosaccharides in the intestine of infants.

Several human milk oligosaccharides inhibit human pathogens in vitro and in animal models. In an infant, the ability of these oligosaccharides to offer protection from enteric pathogens would require that they withstand structural modification as they pass through the alimentary canal or are absorbed and excreted in urine. We investigated the fate of human milk oligosaccharides during transit through the alimentary canal by determining the degree to which breast-fed infants' urine and fecal oligosaccharides resembled those of their mothers' milk. Oligosaccharide profiles of milk from 16 breast-feeding mothers were compared with profiles of stool and urine from their infants. Results were compared with endogenous oligosaccharide profiles obtained from the urine and feces of age-, parity-, and gender-matched formula-fed infants. In all cases, oligosaccharides were extracted, purified, reduced, and separated into acidic and neutral species; the latter were perbenzoylated and subjected to reversed-phase high-performance liquid chromatography. Structures were determined by mass spectrometry after debenzoylation. Among breast-fed infants, concentrations of oligosaccharides were higher in feces than in mothers' milk, and much higher in feces than in urine. Urinary and fecal oligosaccharides from breast-fed infants resembled those in their mothers' milk. Those from formula-fed infants did not resemble human milk oligosaccharides, were found at much lower concentrations, and probably resulted from remodeling of intestinal glycoconjugates or from intestinal bacteria. Most of the human milk oligosaccharides survived transit through the gut, and some were absorbed and then excreted into the urine intact, implying that inhibition of intestinal and urinary pathogens by human milk oligosaccharides is quite likely in breast-fed infants.

Conformational studies of the glycopeptide Ac-Tyr-[Man5GlcNAc-beta-(1-->4)GlcNAc-beta-(1-->Ndelta)]-Asn-Leu-Thr-Se r-OBz and the constituent peptide and oligosaccharide.

Glycopeptides of desired structure can be conveniently prepared by the coupling of reducing oligosaccharides to aspartic acid of peptides via their glycosylamines formed in the presence of saturated aqueous ammonium hydrogen carbonate. The resulting oligosaccharide chains are N-linked to asparagine as in natural glycoproteins, allowing different peptide oligosaccharide combinations to be analysed for conformational effects. In the present paper, a pentapeptide of ovalbumin was coupled to Man5GlcNAc2 oligosaccharide and the glycopeptide and the two parent compounds compared by NMR ROESY experiments and molecular dynamics simulations. Despite the small size of the peptide, conformational effects were observed suggestive of the oligosaccharide stabilising the peptide in solution and of the peptide influencing oligosaccharide conformation. These effects are relevant to the function of glycosylation and the enzymic processing of oligosaccharide chains.

High-performance capillary electrophoresis of sialylated oligosaccharides of human milk.

Oligosaccharides in human milk inhibit enteric pathogens in vitro and in vivo. Neutral milk oligosaccharides vary among individuals and over the course of lactation. To study such variation in the acidic milk oligosaccharides, a sensitive, convenient, quantitative method is needed. High-performance capillary electrophoresis of underivatized acidic oligosaccharides with detection by UV absorbance at 205 nm proved to be sensitive to the femtomole level. Eleven standard oligosaccharides ranging from tri- to nonasaccharide (3'-sialyllactose, 6'-sialyllactose, 3'-sialyllactosamine, 6'-sialyllactosamine, disialyltetraose, 3'-sialyl-3-fucosyllactose, sialyllacto-N-tetraose-a, sialyllacto-N-tetraose-b, sialyllacto-N-neotetraose-c, disialyllacto-N-tetraose, and disialomonofucosyllacto-N-neohexaose) were resolved; baseline resolutions of 3'-sialyllactose, 6'-sialyllactose, and other structural isomers were achieved. Peak areas were linear from 30 to 2000 pg and were reproducible with a coefficient of variation between 4 and 9%. There was no evidence of quantitative interference of one oligosaccharide with another. In studies using pooled human milk, addition of increasing amounts of authentic standard oligosaccharides produced the expected positive increments in detected values, indicating quantitative recovery without interference by other milk components. The identities of the major sialylated acidic oligosaccharides of pooled human milk agreed with the results of previous studies employing other analytical methods. Comparison of oligosaccharide profiles of milk samples from different donors revealed extensive variation, especially in the structural isomers of sialyllacto-N-tetraose. This sensitive, highly reproducible method requires only simple sample workup and is useful in defining variations in human milk acidic oligosaccharides and investigating their possible relationship with diseases of infants.

Milk oligosaccharide profiles by reversed-phase HPLC of their perbenzoylated derivatives.

Human milk is rich in oligosaccharides, some of which inhibit toxins and pathogens involved in diseases of infants. To investigate qualitative and quantitative individual variation of human milk oligosaccharides, a sensitive method for routine identification and quantification of intact milk oligosaccharides was developed and applied to milk samples from 50 donors. The isolated, reduced neutral oligosaccharide fractions were perbenzoylated, resolved by reversed-phase HPLC, and detected at 229 nm. This method resolves most structural isomers and does not require stringent removal of lactose. Peaks were detected at the low nanogram (pmol) level and peak areas were linear from 1 to 1000 micrograms for a standard oligosaccharide. Oligosaccharide samples equivalent to 1 microliter of human milk give optimum chromatographic separation and resolution. The method gives quantitative results comparable to those obtained with classic total sugar analyses, and has an average coefficient of variation of 13%. The 12 major peaks in human milk coeluted with authentic oligosaccharide standards ranging from tri- to octasaccharides, and their identities were confirmed by mass spectrometry. Significant individual variation exists in oligosaccharide profiles; almost 70% of samples contained 2'-fucosyllactose and lacto-N-fucopentaose I as the major oligosaccharides; for the remainder, the major oligosaccharides were 3-fucosytlactose and lacto-N-fucopentaose-II or lacto-N-fucopentaose-III. This method can be used to investigate the extent and biological significance of oligosaccharide variation in human milk.

Mammalian alpha-mannosidases--multiple forms but a common purpose?

Previously, alpha-mannosidases were classified as enzymes that process newly formed N-glycans or degrade mature glycoproteins. In this review, we suggest that two endoplasmic reticulum (ER) alpha-mannosidases, previously assigned processing roles, have important catabolic activities. Based on new evidence, we propose that the ER/cytosolic mannosidase is involved in the degradation of dolichol intermediates that are not needed for protein glycosylation, whereas the soluble form of Man9-mannosidase is responsible for the degradation of glycans on defective or malfolded proteins that are specifically retained and broken down in the ER. The degradation of oligosaccharides derived from dolichol intermediates by ER/cytosolic mannosidase now explains why cats and cattle with alpha-mannosidosis store and excrete some unexpected oligosaccharides containing only one GlcNAc residue. Similarly, the action of ER/cytosolic mannosidase, followed by the action of the recently described human lysosomal alpha(1 --> 6)-mannosidase, together explain why alpha-mannosidosis patients store and excrete large amounts of oligosaccharides that resemble biosynthetic intermediates, rather than partially degraded glycans. The relative contributions of the lysosomal and extra-lysosomal catabolic pathways can be derived by comparing the ratio of trisaccharide Man beta (1 --> 4)GlcNAc beta (1 --> 4)GlcNAc to disaccharide Man beta (1 --> 4)GlcNAc accumulated in tissues from goats with beta-mannosidosis. A similar determination in human beta-mannosidosis patients is not possible because the same intermediate, Man beta (1 --> 4)-GlcNAc is a product of both pathways. Based on inhibitor studies with pyranose and furanose analogues, alpha-mannosidases may be divided into two groups. Those in Class 1 are (1 --> 2)-specific enzymes like Golgi mannosidase I, whereas those in Class 2, like lysosomal alpha-mannosidase, can hydrolyse (1 --> 2), (1 --> 3) and (1 --> 6) linkages. A similar classification has recently been derived by others from protein sequence homologies. Based on this new classification of the alpha-mannosidases, it is possible to speculate about their probable evolution from two primordial genes. The first would have been a Class 1 ER enzyme involved in the degradation of glycans on incompletely assembled or malfolded glycoproteins. The second would have been a Class 2 lysosomal enzyme responsible for turnover. Later, other alpha-mannosidases, with new processing or catabolic functions, would have developed from these, by loss or gain of critical insertion or retention sequences, to yield the full complement of alpha-mannosidases known today.

Complexity of ethanolamine phosphate addition in the biosynthesis of glycosylphosphatidylinositol anchors in mammalian cells.

Biosynthetic intermediates for the mammalian glycosylphosphatidylinositol (GPI) anchor have been described. The earliest GPI anchor precursor is N-acetylglucosaminylphosphatidylinositol, which is deacetylated to give glucosaminylphosphatidylinositol. This is followed by fatty acylation of the inositol ring, sequential addition of mannose residues donated by dolichyl mannosyl phosphate, and finally addition of ethanolamine phosphate. Here, we show that the final steps of GPI anchor biosynthesis are more complex than we have previously reported. Six distinct GPI anchor precursors were found to contain at least 1 ethanolamine phosphate residue. The headgroups of these glycolipids were purified and analyzed by a combination of Bio-Gel P4 chromatography and high resolution thin-layer chromatography. The sizes of neutral glycans were determined following HF dephosphorylation. The position of the ethanolamine phosphate residue was inferred from results of alpha-mannosidase treatment. Finally, the number of negative charges on the headgroups were determined by Mono Q chromatography. Our results show that the addition of ethanolamine phosphate is controlled by at least two different genes. Thus, the class F mutant, though unable to add ethanolamine phosphate to the third mannose residue, does incorporate ethanolamine phosphate into the first and second mannose residues. Only the wild type cells are capable of incorporating ethanolamine phosphate into the third mannose residue. Furthermore, the GPI core contains up to 3 ethanolamine phosphate residues. These results should facilitate the elucidation of the biochemical defects in paroxysmal nocturnal hemoglobinuria.

Ligand recognition by purified human mannose receptor.

In this work we examine the carbohydrate binding properties of human placental mannose receptor (HMR) using a rapid and sensitive enzyme-linked immunosorbent microplate assay. The assay is based on the inhibition of binding of highly purified receptor to yeast mannan-coated 96-well plates. The specificity of ligand binding was inferred from the potency of different saccharides in blocking HMR binding to the mannan-coated wells. The relative inhibitory potency of monosaccharides was L-Fuc greater than D-Man greater than D-Glc greater than D-GlcNAc greater than Man-6-P much greater than D-Gal much greater than L-Rha much greater than GalNAc. The inhibitory potency of mannose increased by two orders of magnitude when linear oligomers were used. Oligomers containing alpha-1-3- and alpha-1-6-linked mannose residues were more inhibitory than those containing alpha-1-2- and alpha-1-4-linked mannoses. Linear or branched oligomannosides larger than three units did not have a significant influence on their inhibitory potency; rather, potency was found to decrease in comparison with oligomannosides with three units. Compared to linear oligomers, inhibition of binding was the best using branched mannose oligosaccharides, alpha-D-Man-bovine serum albumin conjugates, or mannan. A model is discussed in which branched ligand is bound to spatially distinct sites on the HMR.

Inhibition of glycosylphosphatidylinositol anchor formation by mannosamine.

Many eucaryotic cell surface proteins are anchored to the plasma membrane via a glycosylphosphatidylinositol (GPI), of which the core region is highly conserved from protozoa to mammalian cells. Previous studies (Lisanti, M. P., Field, M. C., Caras, I. W., Menon, A. K., and Rodiguez-Boulan, E. (1991) EMBO J. 10, 1969-1977) showed that mannosamine blocked the expression of a recombinant GPI-anchored protein in Madin-Darby canine kidney cells and converted this protein to an unpolarized secretory product. In the present study, we examined the effect of mannosamine on the formation of the glycan portion of the GPI anchor precursors. This amino sugar inhibited the incorporation of mannose into the glycan portion, and the inhibition was dose-dependent. Mannosamine was shown to be incorporated into the glycan as mannosamine, probably mostly in the second mannose position and thereby to block the further addition of mannose and other anchor components. The products formed in the presence of this drug were characterized by gel filtration and high resolution TLC both before and after deamination with nitrous acid and dephosphorylation by HF. Galactosamine and trehalosamine were inactive in this system, whereas glucosamine also inhibited mannose incorporation into GPI intermediates.

Substrate specificity of the bovine and feline neutral alpha-mannosidases.

Neutral alpha-mannosidases were prepared from bovine and cat liver. The activities were distinguished from lysosomal and Golgi alpha-mannosidases by their neutral pH optima, relatively low Km for their synthetic substrate p-nitrophenyl alpha-D-mannoside, inhibition by Zn2+ and absence of inhibition by Co2+, EDTA, low concentrations of swainsonine, or deoxymannojirimycin. The cytosolic alpha-mannosidases were not retained by concanavalin A-Sepharose. They were able to degrade efficiently a variety of oligosaccharides with structures corresponding to certain high-mannose glycans or the oligomannosyl parts of hybrid and complex glycans. However, unlike lysosomal alpha-mannosidases from the same species these enzymes were not able to degrade Man9GlcNAc2 efficiently, and the bovine neutral alpha-mannosidase was not able to degrade a hexasaccharide with a structure analogous to Man5GlcNAc2-PP-dolichol. Sharp differences were noted for the bovine and cat enzymes with regard to the specificity of degradation. The bovine neutral alpha-mannosidase degraded the substrates by defined pathways, but the cat neutral alpha-mannosidase often produced complex mixtures of products, especially from the larger oligosaccharides. Therefore the bovine enzyme resembled the rat and human cytosolic alpha-mannosidases, but the cat enzyme did not. The bovine and cat neutral alpha-mannosidases, unlike the corresponding lysosomal activities, did not show specificity for the hydrolysis of the (1----3)- and (1----6)-linked mannose residues in the N-linked glycan pentasaccharide core.

A human lysosomal alpha(1----6)-mannosidase active on the branched trimannosyl core of complex glycans.

Normal human fibroblasts and fibroblasts from a patient with alpha-mannosidosis were grown in the presence or absence of 100 microM swainsonine for 7 days. Accumulated oligosaccharides were isolated and analysed by high performance liquid chromatography (HPLC) and methylation analysis. Man alpha 1----3Man beta 1----4GlcNAc and Man alpha 1----2Man alpha 1----3-Man beta 1----4GlcNAc (where Man is D-mannose and GlcNAc is N-acetyl-D-glucosamine) comprised greater than 80% of the total oligosaccharides in untreated mannosidosis cells. However, Man alpha 1----6[Man alpha 1----3]Man beta 1----4GlcNAc was the major Man3GlcNAc isomer present after 7 days of swainsonine treatment. No mannose-containing oligosaccharides were detected in control fibroblasts in the absence of swainsonine but, in its presence, oligosaccharides containing 2-9 mannose residues accumulated. Man alpha 1----6[Man alpha 1----3]-Man alpha 1----6[Man alpha 1----3]Man beta 1----4GlcNAc and Man alpha 1----6-[Man alpha 1----3]Man beta 1----4GlcNAc were the major components (67%). Surprisingly, Man alpha 1----3Man beta 1----4GlcNAc was only observed in swainsonine-treated control cells during the recovery period after removal of swainsonine. These studies suggest the presence of a second lysosomal alpha-mannosidase activity which is unaffected in genetic alpha-mannosidosis, but is inhibited by swainsonine. This enzyme would cleave the alpha(1----6)-linked mannose residue from branched Man3GlcNAc to form Man alpha 1----3Man beta 1----4GlcNAc. To confirm this hypothesis, fractions from alpha-mannosidosis and control fibroblasts that bound to concanavalin A (ConA)-Sepharose and were eluted with 0.5 M alpha-methyl mannoside were incubated at pH 4.0 with Man alpha 1----6[Man alpha 1----3]Man beta 1----4-GlcNAc. As anticipated, Man alpha 1----3Man beta 1----4GlcNAc was the sole product using enzyme from mannosidosis fibroblasts, while the major product from control fibroblasts was Man alpha 1----6Man beta 1----4GlcNAc. This confirmed the presence of a swainsonine-inhibitable alpha(1----6)-mannosidase activity unaffected by the disease. The differing substrate specificities of the alpha(1----6)-mannosidase and the major lysosomal alpha-mannosidase indicate that the alpha(1----6)-mannosidase plays an important role in the generation of the oligosaccharides accumulated in alpha-mannosidosis patients.